mTOR (mammalian target of rapamycin) was first identified in 1993 as the protein target of rapamycin (TOR2) and an essential phosphatidylinositol 3-kinase (PI3K) homolog (Kunz et al., 1993). mTOR is a serine/threonine kinase and regulates protein translation and progression from G1 to S phase of the cell cycle (Kuruvilla et al., 1999). mTOR is essential for cell survival because it promotes protein translation, cell proliferation, and growth.
MTOR gene knock-out leads to cell arrest at G1 phase and halts cell proliferation and growth (Murakami et al., 2004; Zhang et al., 2000; Kunz et al., 1993; Brown et al., 1994; Kuruvilla, et al., 1999). The mTOR inhibitor rapamycin (first identified in 1975 by Vezina et al. as an antifungal antibiotic isolated from Streptomyces hydroscopicus) and certain of its analogs (also known as rapalogs) have been FDA approved and used as a treatment for tumors (such as renal cell carcinoma, mantle cell lymphoma, and breast cancer), as these compounds inhibit mTOR-mediated phosphorylation and subsequent downstream cell growth and proliferation.
With the growing utilization of rapid sequencing technology, many human tumors (5-10%) have been found to contain somatic mutations in the MTOR gene (Grabiner et al., 2014). There are 463 reported somatic mutations in the MTOR gene in the COSMIC (Catalogue of Somatic Mutations in Cancer) database. The use of rapid sequencing technology such as Whole Genome Sequencing (WGS) or Next Generation Sequencing (NGS) is expected to permit identification of genetic variants that may alter treatment of genetically complex diseases, thereby promoting personalized medicine. However, the massive genome information lacks the support of interpretation because the phenotypic effects of the gene mutations are not known. The current knowledge of mutations in the MTOR gene is mostly limited to some associations with increased mTOR activity. Certain MTOR gene mutations are known to be associated with an increase in mTOR activity, thereby increasing the rate of tumor growth. (Grabiner et al., 2014). Rapamycin treatment is a cancer therapy that is targeted to reduce the mTOR activity. Information relating to specific MTOR gene mutations that confer resistance to rapamycin treatment is lacking. Additionally, ATP-competitive inhibitors of mTOR have been developed and are currently in clinical trials (Thoreen et al., 2009; Schenone et al., 2011). However, mutations in the MTOR gene can also decrease the efficacy of these newer medications (Wu et al., 2015; Rodrik-Outmezguine et al., 2016).
Several methods are available that purportedly determine mTOR activity. One such method involves detection of the phosphorylation status of the P70 S6 kinase (AlphaScreen SureFire assay, PerkinElmer). Another method relates to an ELISA that measures the kinase activity of mTOR immunoprecipitated from cell lysates (K-LISA mTOR Activity Kit, EMD Millipore). However, these two methods have significant drawbacks and do not accurately measure the effectiveness of a particular mTOR inhibitor as it related to the downstream function of mTOR activity, which is multifaceted.
Another known method includes the FKBP12-FRB (FKBP12-Rapamycin binding domain of mTOR) split luciferase assay that measures emission of light signals upon binding of FKBP12 to FRB domain when rapamycin is active in reducing mTOR activity (Luker et al., 2004; Dixon et al., 2016). However, this particular method is hindered by the use of a small portion of mTOR, thus precluding its effective assessment of the entire MTOR gene. In other words, this method precludes an effective assessment of all MTOR genetic variants and is limited to evaluating the ability of rapamycin to bind to a small portion of mTOR. These methods represent an inaccurate depiction of the effectiveness of whether a particular inhibitor can reduce mTOR activity, thereby inhibiting the tumor growth.
Accordingly, there is a continuing need to develop a rapid cell-based assay that measures mTOR activity and the effectiveness of inhibitors against alterations in mTOR activity due to somatic mutations in the MTOR gene.